Mammalian cells are the most suitable expression system for obtaining different types of recombinant proteins, particularly those proteins intended for therapeutic applications. Among the most effective expression vectors for incorporating a gene of interest in a cell for the purpose of expressing a protein of interest are alphavirus-based vectors. Alphaviral expression vectors have been developed from different types of alphavirus, including Sindbis virus (SIN), Semliki Forest Virus (SFV) and Venezuelan equine encephalitis (VEE) virus.
The alphavirus replicon contains at its 5′ end an open reading frame encoding viral replicase (Rep) which is translated when viral RNA is transfected into eukaryotic cells. Rep is expressed as a polyprotein which is subsequently processed into four subunits (nsps 1 to 4). Unprocessed Rep can copy the RNA vector into negative-strand RNA, a process that only takes place during the first 3 to 4 hours after transfection or infection. Once processed, the Rep will use the negative-strand RNA as a template for synthesizing more replicon molecules. Processed Rep can also recognize an internal sequence in the negative-strand RNA, or subgenomic promoter, from which it will synthesize a subgenomic positive-strand RNA corresponding to the 3′ end of the replicon. This subgenomic RNA will be translated to produce the heterologous protein in large amounts.
Normally, alphavirus vectors are based on RNA replicons in which the structural genes have been substituted with a heterologous gene. However, the replication of most alphaviral vectors is cytopathic, so said vectors do not allow obtaining long-lasting expression of the gene of interest. To solve that problem, several groups have identified a series of mutations in alphavirus replicase which can convert these cytopathic viral vectors into non-cytopathic viral vectors, allowing a more long-lasting expression of the recombinant products expressed by the viral vector. These studies have led to the generation of different alphavirus-derived non-cytopathic vectors.
A non-cytopathic mutant isolated from SIN containing a single amino acid change (P for L) in position 726 in nsp2 (SIN P726L vector in nsp2) showed Rep hyperprocessing (Frolov et al., 1999, J. Virol. 73: 3854-65). This mutant was capable of efficiently establishing continuous replication in BHK cells. This non-cytopathic SIN vector has been widely used in vitro as it is capable of providing long-lasting transgene expression with good stability levels and expression levels that were about 4% of those obtained with the original SIN vector (Agapov et al., 1998, Proc. Natl. Acad. Sci. USA. 95: 12989-94). Nevertheless, although said vector is not cytopathic, it lacks the capacity to generate stable cell lines with high expression levels.
With respect to non-cytopathic SFV mutants described by Perri et al. (Perri et al., 2000, J. Virol. 74: 9802-7), including mutants SF2A (mutation L10T in nsp2) and SF2C (mutation L713P in nsp2), as well as double mutant PD (S259P and R650D in nsp2) described by Lundström et al. (Lundström et al., 2001. Histochem. Cell. Biol. 115: 83-91), although they can express similar or even higher protein levels than those of the wild-type virus (PD mutant), these mutants continue to be cytopathic in all cases and the generation of stable cell lines expressing heterologous proteins based on said viral vectors has not been described.
Patent application WO2008065225 describes a non-cytopathic SFV vector as a result of the presence of mutations R649H/P718T in the replicase nsp2 subunit. Said vector allows obtaining cell lines capable of constitutively and stably expressing the gene of interest by means of culturing in the presence of an antibiotic the resistance gene of which is incorporated in the alphaviral vector (Casales et al. 2008. Virology. 376:242-51).
Use of non-cytopathic replicon-based viral vectors is associated with the drawback that mutations can accumulate in the gene of interest because the viral replicase, which lacks error correcting activity, is constantly copying viral RNA. A second drawback of these systems is the fact that they do not allow the expression of toxic proteins for the cell since the expression occurs constitutively. Finally, a decrease in expression levels of the gene of interest is often observed in these systems, which can in part be due to the fact that adaptation of the cells to grow in the presence of the antibiotic, necessary for maintaining expression of the alphaviral replicon, can cause the cells to require a smaller amount of replicon to survive. These problems could theoretically be solved by means of designing a viral vector integrated in the cell genome and in which expression of the alphaviral replicon can be regulable by means of inducible promoters or other systems (WO9738087, WO2000006205 and WO2001081553). However, there is no evidence that such vectors have allowed obtaining high expression of the proteins of interest in a sustainable manner over time. Use of DNA sequences complementary to the RNA sequences making up the alphaviral replicon has allowed designing alphaviral vectors that allow integrating the gene of interest in the cell genome, thereby favoring obtaining a cell line capable of stably expressing the gene of interest without the need to add an antibiotic. However, cell lines obtained by means of these vectors often show great variability as regards expression levels of the proteins of interest, as well as, if appropriate, inducibility of expression of these proteins, probably due to differences in the integration site and the number of integrations in the cell genome, so isolating and selecting those clones with optimal characteristics is necessary.
Therefore there is a need to provide an expression system for expressing genes of interest in mammalian cells which allows stably expressing said genes in large amounts, such that it is suitable for the large-scale production of recombinant proteins.